Abstract Tuberculosis remains a major cause of morbidity and mortality worldwide, and is the leading cause of death due to a single infectious agent, Mycobacterium tuberculosis. Most countries still vaccinate newborns with the only licensed vaccine against tuberculosis, Bacille Calmette Guerin (BCG) an attenuated version of Mycobacterium bovis. The vaccine was developed over a century ago and, although widely used, has limited efficacy. BCG delivered intradermally (ID) does provide protection against the worst manifestations of tuberculosis in infants but has variable (0-80%) protection against pulmonary TB. Our recent data in non-human primates (rhesus macaques) demonstrate that BCG delivered by the intravenous (IV) route provides sterilizing or near sterilizing protection in 90% of macaques (>10,000 fold reduction in bacterial burden overall), while ID BCG provides little protection in this model. However, delivery of even an attenuated live vaccine poses potential safety issues, particularly in immune compromised subjects. In this proposal, we aim to construct BCG strains that are self-inactivating, using several innovative molecular approaches. Such strains would persist only for a short time in vivo, and would likely die by different mechanisms. We will construct and characterize the BCG self-inactivating strains in vitro (Aim 1), assess persistence and immunogenicity in a murine model, and then determine persistence, immunogenicity and protection of a subset of BCG strains in a susceptible non-human primate model (rhesus macaques). Published data indicate that the robust protection seen by IV BCG in macaques is not recapitulated in mice, which necessitates testing in macaques. We will use sophisticated technology for construction of strains, assessment of immune responses and bacterial burden, and PET CT imaging for tracking protection. This proposal builds on the experience of the Multi-PI team in mycobacterial genetics, immunology, and animal models, with the goal of developing a safer TB vaccine for IV delivery.